Enzymatic carboxylmethylation of calmodulin by protein-O-carboxylmethyl-transferase (PCM; E.C 2.1.1.24) diminishes the ability of calmodulin to subsequently activate phosphodiesterase. A partially purified cAMP phosphodiesterase was purified by chromatography over fluphenazine-Sepharose followed by calmodulin-Sepharose. When this preparation was incubated with PCM and S-adenosylmethionine, a marked inhibition of Ca++-calmodulin stimulation was seen at all concentrations of calmodulin. In contrast, prior carboxylmethylation of calmodulin only reduced the stimulation in phosphodiesterase activity at subsaturating concentrations of calmodulin. Thus, carboxylmethylation may regulate the activity of cyclic nucleotide phosphodiesterase by directly altering the enzyme as well as by carboxylmethylating calmodulin.